The invention relates to liquid chromatography apparatus and to a method of optimising solvent compositions for the analysis of a sample by liquid chromatography.
Chromatography has been used for many years to separate and measure the concentration of the constituents of complex mixtures. To analyze a sample in a liquid chromatograph apparatus an unknown sample is injected into a separating column together with a solvent (or mobile phase) or mixture of solvents. A detector at the far end of the column detects the presence of the constituents as they emerge or elute from the column. A chromatographer uses a plot of the detector output against time, known as a chromatogram, to analyze the unknown sample.
For example, an analyst may wish to know the concentration of a given pesticide in water or a given pharmaceutical drug in the bloodstream of a patient. In that case a known volume of the water or of the blood would be injected into a separating column and the constituent parts including the pesticide or the drug would be separately eluted and can be detected. By knowing when the particular constituents should elute their presence can be detected and quantative measurement may be made on the peaks.
Such a method of operation is very useful when the constituents of the sample are known. However, when the sample is an unknown, the chromatogram produced is more difficult to interpret. Further, research has shown that the separation of unknowns into their constituent parts in a liquid chromatograph is not always possible with a given multi-component solvent composition. As a result, analysts have varied the concentrations of the constituents of the solvent and have used solvents having up to four different major constituents enabling the separation of more kinds of unknowns into their component parts, provided a proper solvent composition can be selected. However, if an improper solvent composition is used, two or more components may co-elute, thereby producing a chromatogram with insufficient resolution. Thus chromatographers are left with the very time-consuming problem of randomly experimenting with the different solvent compositions and evaluating the chromatogram for each such experiment to determine which one is best. Typically, a chromatographer may perform a large number of experiments on a given unknown sample, each experiment being done with a different solvent composition in the column. A chromatogram is produced from each experiment. For a typical unknown, the chromatographer may perform many experiments before he discovers a set of conditions which are acceptable but, even so, perhaps not ideal for his purposes. Some of the chromatograms are easily discarded as being unusable but evaluating the remaining chromatograms to determine which is the most usable is very difficult. This task becomes even more difficult as the number of experiments increases as is required when three or four solvents are used in the column. Without a system and protocol it becomes highly unlikely that an optimum separation will be achieved by trial and error.
This problem has been addressed in a paper by Haleem J. Issaq entitled Computer-assisted HPLC which was published in American Laboratory, February 1983, pp 41-46. The paper describes a method based on statistical calculations whereby peak pair resolutions in 5 to 10 chromatographic runs using a combination of two or three pure or mixed solvents are plotted versus mobile phase composition. If no peak crossover takes place the resolution between each pair is used. If peak reversal does occur, the resolution between all peaks is calculated and used in determining the optimum mobile phase. The resulting overlapping resolution mapping (ORM) plots of each pair are then generated by the computer and indicate the regions where resolutions are above a level predetermined by the analyst. The union of all peak pair ORM plots, handled automatically by the computers, will give one plot indicating the region where all peak pair resolutions are above a predetermined level.
Since resolution versus mobile phase composition is used to generate the ORM plots, the analyst can program the computer to predict and print in the form of a table the peak pair resolutions for any desired mobile phase composition.
Retention times may be predicted if a plot of retention time, instead of resolution, is plotted versus mobile phase composition for each component in such mobile phase. This will be of value in showing how long it will take for the last component to elute from the column. In cases where resolution and not retention time is predicted, the analyst does not know how long the chromatographic run will take.